Enzymes are believed to utilize binding energy from the interaction of nonreacting groups of specific substrates with the active site to bring about rate increases. As with substrate binding, covalent bond rearrangement steps in an enzyme reaction sequence also involve energy changes and the question arises at to the role the covalent transformations play in the process of catalysis. It is the purpose of this proposal to concentrate on those steps in the catalytic sequence that involve covalent changes and, in particular, to provide a thermodynamic characterization of the covalent and noncovalent contributions. Free energy data at pH 7 will be sought for the unfolding of alpha- chymotrypsin (alpha-Ct) as well as for covalent enzyme-substrate species such as o-hydroxphenylmethanesulfonyl-chymotrypsin and furoyl-chymotrypsin. The known free energy diagrams for the alpha- chymotrypsin mediated hydrolysis of the substrates which give rise to these intermediates will be combined with the corresponding unfolding free energy data in order to evaluate the free energy change for covalent bond rearrangement in the enzyme mediated reaction. A complete enthalpy diagram for the reaction of alpha- Ct with o-hydroxyphenylmethanesulfonic acid sultone will be sought to complement the free energy diagram and permit evaluation of entropic effects in the noncovalent and covalent contributions to the reaction sequence. The methods for evaluation of covalent and noncovalent contributions to the free energy-reaction coordinate profile of enzyme mediated reactions will be applied to other enzymes, such as acid phosphatase and Staph. aureus penicillinase. In the case of penicillinase, the effect of substrate ring strain on the enzyme mediated decomposition of quinicillin will be investigated in terms of the reaction sequence and covalent and noncovalent contributions to the thermodynamics.